Abstract T cells are central components of the adaptive immune response. In order to protect the host from a variety of different pathogens, these cells must be activated by the binding of foreign peptide antigens to their receptors, the T cell receptors (TCRs), in the context of the major histocompatibility complex protein (MHC). Previous studies have demonstrated that the depolarization of human leukemic T cells dampens TCR stimulation- induced calcium signaling through non-voltage gated Ca2+ channels in the cell, a canonical marker of T cell activation. However, a fundamental understanding of how membrane depolarization regulates the activation state of a T cell is unclear. Due to the size of the TCR-CD3 complex and its location in the membrane, it has been challenging to obtain structural information about the whole complex. It is known that there are charged residues in the transmembrane (TM) region and polybasic residues in the cytoplasmic domain that might play a role in complex assembly and regulating immunomodulatory tyrosine availability for phosphorylation by kinases in the cytoplasm, respectively. Previous work by co-sponsor Bezanilla has illustrated the ability of charged residues in TM proteins to move upon changes in membrane voltage in a manner that is crucial to cellular function. The main question this proposal aims to address is how depolarization of the T cell membrane can regulate the conformation and function of the TCR-CD3 complex. We will use two novel free-standing silicon nanomaterials that the applicant recently developed in the Tian laboratory to optically depolarize the membranes of T cells. We will then perform Frster resonance energy transfer imaging to examine how CD3 cytoplasmic subunits move relative to the membrane and how TCR-CD3 TM subunits move relative to the TCR extracellular domains, upon nanomaterial induced depolarization and stimulation with various purified peptide- MHC ligands produced in the Adams laboratory. We will also study changes in the expression and phosphorylation status of proximal and downstream T cell signaling proteins in the Adams laboratory upon membrane depolarization and TCR stimulation. The results of this work will provide biophysical and functional insight into how plasma membrane potential can regulate T cell activation with potential implications for therapeutic strategies in autoimmunity diseases.